PROJECT SUMMARY Specific targeting of the Ataxia Telangiectasia and Rad3-related kinase (ATR) represents an emerging strategy to treat a broad spectrum of cancers, most notably those that currently lack effective treatments. Suppression of ATR selectively kills cells subjected to oncogenic stress, alternative lengthening of telomeres (ALT) or loss of double strand break (DSB) repair mechanisms (ATM, BRCA1, or BRCA2 deficiency). Indeed, levels of ATR suppression that eliminate such cancers do so with minimal toxicity to tissues under normal proliferative control, including those that are the most sensitive to traditional chemotherapeutics such as the bone marrow and intestine. Therefore, ATR inhibitors provide a new and effective treatment for cancer, one that causes fewer side effects than conventional chemotherapies. Atrin Pharmaceuticals has synthesized a novel series of small molecules that inhibit ATR at low nanomolar concentrations in cultured cells. These compounds have the highest known potency for inhibiting ATR and maintain >833-fold lower in vivo activity towards other kinases of the same family (ATM, DNA-PKcs and mTOR), which are substantially off-targeted by all previously reported ATR inhibitors. Herein, we propose to complete our preliminary evaluation of Atrin?s ATR inhibitor series by: 1) further defining the mechanism of action and enhancing the pharmacologic properties of the ATRN series, and 2) exploring the ability of the ATRN series to kill cancers in combination with conventional therapies. The ultimate goal of this proposed research is to produce a uniquely specific small-molecule inhibitor of ATR that can be applied in the clinic.